Photochemical and biological consequences of the photocrosslinking of lysozyme

LUIS DORADO; V. REY; A.E. LEDESMA; BEATRIZ LOPEZ DE MISHIMA; CARLA E. GIACOMELLI; CLAUDIO BORSARELLI

Salta

Congreso; Latin American Protein Society Meeting (LAPSM).; 2010

Protein oxidation results in severe losses of biological functionality due to changes on the structure and/or conformation of the native protein. Depending on the type of oxidative agent and oxidation degree, the damage of the protein can result in peptide bond breakdown, aminoacid residue modification, intra- and/or crosslinking, etc. Lysozyme (LYZ) or muramidase, a 14.4 kD antimicrobial protein, is very abundant in a number of secretions, such as tears, saliva, human milk, mucus, and in egg white. LYZ has been used as preservative in foods. However, LYZ in combination with a sensitizer, such as riboflavin, and visible light is unstable in solution. In the present work, we have studied the formation of covalent oligomeric species of LYZ by action of protein tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine (Rubpy) in the presence of ammonium persulfate (APS). Several spectroscopic techniques (stationary and dynamic UV/Vis absorption and fluorescence) together with the analysis of SDS-PAGE were used to characterize the photocrosslinked products of LYZ. The formation of the covalent oligomeric species (dimer, trimer, …n-mer) was proportional with the irradiation time. The structural changes were parallel with the quenching of the intrinsic tryptophan (Trp) emission at 340 nm together with the increment of the typical di-tyrosine (di-Tyr) emission at 405 nm. Therefore, the Trp and Tyr residues participate in the photoinduced formation of both oxidation products and/or LYZ oligomeric species. The antimicrobial activity of these species was compared with that of native LYZ.